purpose. To investigate in impression cytology (IC) specimens the expression of
inflammatory and apoptosis-related markers by conjunctival epithelial
cells from patients with dry eye as a rationale for treatment with
topical cyclosporine.

methods. Immunologic anomalies were identified at baseline, before treatment
with the masked medication, in a homogeneous series of patients with
dry eye syndrome, who were enrolled in a large European multicenter
clinical trial (Cyclosporin A Dry Eye Study; Allergan, Irvine, CA). IC
specimens were collected in 243 patients with moderate to severe
keratoconjunctivitis sicca (KCS), with or without Sjögren’s
syndrome (SS). Fifty normal subjects were separately examined to
provide normal control values. Specimens were analyzed in a masked
manner by flow cytometry, using antibodies directed to markers of the
immune system and/or apoptotic pathway: HLA DR, CD40, CD40 ligand, Fas,
and APO2.7. Levels of expression were quantified, and results were
compared with those obtained in the 50 normal patients.

results. One hundred sixty-nine specimens were successfully interpreted at
baseline, including 41% from patients with SS. A highly significant
increase of HLA DR expression by conjunctival cells was found in
KCS-affected eyes compared with normal eyes, which did not express this
marker or did so very weakly. HLA DR expression in eyes with SS was
significantly higher than in KCS-affected eyes without SS. Fas and
APO2.7 were found at low levels in all normal and KCS-affected eyes.
CD40 and CD40 ligand expressions were significantly increased in eyes
with KCS compared with normal eyes. HLA DR, CD40 and Fas were found at
significantly higher levels in the SS group than in the non-SS group.

conclusions. Conjunctival cells from patients with dry eye with moderate to severe
KCS, with or without SS, overexpress inflammatory and apoptosis-related
markers. Whether inflammation is a primary phenomenon in KCS or is the
consequence of repetitive abrasion of the ocular surface after tear
film deficiency remains to be determined. These data, nevertheless,
support the use of immunomodulatory and/or anti-inflammatory drugs in
the treatment of patients with KCS.

Dry eye disease or keratoconjunctivitis sicca (KCS) is one of the
most frequently encountered categories of ocular morbidity in the
United States, with as many as 4.3 million persons above age 65 having
symptoms of dry eye often or all the time.1 The
tear-deficient dry eye is characterized primarily by a deficiency in
tear production and includes KCS associated with Sjögren’s
syndrome (SS), an autoimmune disease of the lachrymal gland and ocular
surface. The histopathologic changes of the lachrymal gland in patients
with SS therefore consist of lymphocytic infiltration leading to
atrophy and destruction of glandular function.2

Most cases of lachrymal gland insufficiency, however, cannot be
attributed to this syndrome. Williamson et al.3 and Damato
et al.4 also demonstrated the presence of lymphocytic
infiltrates in the lachrymal glands of patients who had KCS but not SS
and suggested that atrophy of the lachrymal gland that occurs with
senescence represents a chronic, progressive inflammatory process. A
mechanism for this inflammatory process in patients without SS recently
has been proposed that describes alterations in membrane trafficking of
acinar cells leading to the expression of major histocompatibility
complex class (MHC) II molecules capable of triggering an autoimmune
response.5 Even in non-SS–affected dry eyes, an
inflammatory reaction has been demonstrated that could be hypothesized
to result from chronic ocular surface dryness and degeneration after
tear deficiency. A strong expression of class II antigens by
conjunctival epithelial cells has thus been assessed in KCS by
immunocytologic techniques, using impression cytology
(IC)678 or brush cytology.9

Immune-based inflammation is a common feature of many ocular surface
syndromes including dry eye disease. A strong relationship has also
been proposed between inflammatory pathways and apoptosis, which
directly affects epithelial turnover.1011 Increased
expression of apoptotic modulators in ocular surface models of wound
healing include the Fas (CD95) system (Fas and Fas ligand) and the CD40
system (CD40 and CD40 ligand), both as membrane-bound and soluble
proteins.121314 Fas and CD40 antigens are two membrane
receptors that belong to the tumor necrosis factor (TNF)/nerve-growth
factor receptor family and mediate apoptosis when bound respectively to
Fas ligand and CD40 ligand (two members of the TNF family).
Fas15 and CD4016 overexpressions were shown
in conjunctival IC specimens from patients with KCS and various
inflammatory ocular surface disorders. Demonstration of increased
expression of immune markers and antigens of apoptotic pathways in
patients with KCS, with and without SS, would therefore support the
role of ocular surface inflammation in the pathogenesis of KCS and the
use of immunomodulatory and/or anti-inflammatory drugs in its
treatment.

The objective of this study was thus to assay inflammatory and
apoptosis-related markers in conjunctival cells from patients with KCS
enrolled in a large European multicenter trial on cyclosporine
ophthalmic emulsion (Cyclosporin A; Allergan, Irvine, CA) in the
treatment of moderate to severe KCS, using a flow cytometry technique
previously validated in IC specimens.715 This study was
undertaken to confirm the existence of inflammatory and
apoptosis-related markers in ocular surface cells of patients with KCS
and to determine whether treatment with cyclosporine could induce
changes in these markers. IC was performed at baseline, 3, 6, and 12
months. The present study reports investigations on five markers
involved in apoptosis and immune reactions that were previously shown
to be overexpressed in ocular surface diseases.67891516 Expressions of Fas, CD40, CD40 ligand, the apoptotic marker APO2.7 and
HLA DR class II antigen, as the main standard of inflammation, were
therefore analyzed at baseline, before cyclosporine or vehicle
treatment, to assess the baseline inflammatory status of the ocular
surface in a large series of patients with moderate to severe KCS.

Materials and Methods

Study Design

A multicenter, double-masked, randomized, vehicle-controlled,
parallel-group study of the safety and efficacy of Cyclosporin A 0.05%
and 0.1% ophthalmic emulsions used twice daily for up to 2 years in
patients with moderate to severe KCS was designed by Allergan (protocol
192371-501-03). Qualified patients entered a 2-week run-in phase during
which they were instructed to administer only ophthalmic lubricant
(Refresh; Allergan) to each eye as needed daily (day −14 to day 0).
The qualification, or baseline, examination (day 0) included
questionnaires to quantify subjective assessments, a Schirmer’s tear
test (with and without anesthesia), visual acuity, biomicroscopy, tear
break-up time, corneal and interpalpebral conjunctival staining
(lissamine green and sodium fluorescein), and intraocular pressure.
Blood samples were obtained to test for SS-related autoantibodies
before randomization. Patients who completed the run-in phase and still
fulfilled the inclusion and exclusion criteria (Table 1) were qualified to enter the masked treatment phase at the
qualification/baseline visit.

At selected centers, IC specimens were collected by the physician on
the qualification-baseline visit (day 0). Specimens were obtained from
the worse eye of patients who met the entry criteria and qualified for
randomization and who had given specific consent for this procedure to
be performed. The worse eye was defined as the one showing the highest
degree of corneal staining, or the lowest Schirmer’s test result when
both eyes had the same corneal scores. If the two criteria were equal
in both eyes, the right eye was chosen for IC. The patients providing
samples for this study were recruited from 29 of 35 centers involved in
the European trial. The conjunctival samples were collected by the
investigators from four countries throughout Europe and shipped to the
Immunohematology Department, Ambroise Paré Hospital (Boulogne,
France) for centralized analyses.

Baseline data presented in this report were analyzed by flow cytometry
on samples gathered from 169 patients. To provide normal reference
values for the tested markers, 100 control eyes from 50 normal subjects
were also examined under similar procedures in independent
investigations performed after ethics committee approval. Patients were
assessed as normal according to history data, complete slit lamp
examination, tear break-up time recording, fluorescein test, and
lissamine green staining. Only patients with absolutely normal criteria
and not having received eye drops for at least 2 months were collected
for normal population analyses.

The laboratory work for this study and the study protocol were
conducted in compliance with the Ethics Committee (CCPPRB) at the
Ambroise Paré Hospital and the relevant Ethics Committees in each
of the participating countries. Written, informed consent was obtained
before enrollment in the study, and patients were given the option to
provide or not provide conjunctival impressions for this study.
Specific additional consent for the impression procedure was obtained
in all cases. This study was conducted in compliance with the
Declaration of Helsinki, Somerset–West amendment, 1996.

Sample Collection and Handling

After the instillation of one drop of topical anesthetic (0.04%
oxybuprocaine), two to three filters 13 × 6.5 mm in size
(polyethersulfone filters, 0.20-μm pores, 13-mm in diameter; Supor;
Gelman Sciences, MI) were applied to neighboring areas of the superior
and superotemporal bulbar conjunctiva without exerting any pressure,
according to previously published procedures.715 Specimens were collected at least 15 minutes after instillation of the
last staining eye drop (i.e., fluorescein and lissamine green), to
avoid any interference with immunofluorescence analyses. Care was taken
to collect IC samples from nonexposed regions of the conjunctiva.
Membranes were removed immediately after contact. Approximately 50% to
70% of the total surface of the filter was to be covered by cells. If
not, investigators were instructed to collect a new specimen from an
adjacent area. All membranes from each eye were immediately dipped into
tubes containing 1.5 ml of cold phosphate-buffered saline (PBS, pH 7.4)
with fixative (0.05% paraformaldehyde, prepared monthly and sent
regularly from the central laboratory to the centers). Tubes were to be
kept at 4°C before impression collection and sent within 2 days to
the Department of Immunohematology, Ambroise Paré Hospital, in
cold-conditioned containers. After reception by the central laboratory,
cells were extracted by gentle agitation for 30 minutes and centrifuged
(1600 rpm, 5 minutes). They were then counted in a Malassez cell before
processing for flow cytometry. The conjunctival samples were processed
up to a week after samples were collected. Because the samples were
kept in fixative solution at 4°C, no major sample degradation was
observed, as assessed elsewhere in repeated flow cytometric analyses
over 4 weeks after collection (data not shown).

Antibodies and Immunofluorescence Procedures

Five sets of antibodies and two corresponding negative controls
were used for assaying: CD95/Fas, the apoptotic marker APO2.7, class II
antigen HLA DR, CD40, and CD40 ligand. One antibody was used for the
direct immunofluorescence procedure for the following two labels:
fluorescein isothiocyanate (FITC)-conjugated mouse IgG1 anti-human CD95
(clone UB2, 1 mg/ml; Immunotech, Marseilles, France), and phycoerythrin
(PE)-conjugated mouse IgG1 anti-human APO2.7 (clone UB2,1 mg/ml,
Immunotech). APO2.7 is a mitochondrial protein reliably expressed by
cells involved in the apoptotic pathway.15 The
FITC/PE-conjugated nonimmune mouse IgG1 was used as a negative isotypic
control for direct immunofluorescence procedure. Two sets of antibodies
were successively used for the indirect immunofluorescence procedure.
The primary antibodies were mouse IgG1 anti-HLA DR α-chain (clone
TAL.1B5, 50 μg/ml; Dako, Copenhagen, Denmark), mouse IgG1 anti-CD40
(clone MAB89, 1 mg/ml; Immunotech), and mouse IgG1 anti-CD40 ligand
(clone TRAP1, 1 mg/ml; Immunotech). FITC-conjugated goat anti-mouse
immunoglobulins was used as the secondary antibody for all the assays
(Dako). The nonimmune mouse IgG1 was used as a negative isotypic
control for the indirect immunofluorescence procedure (Dako).

Antibodies were used in a 1:50 dilution in 1% bovine serum albumin
containing PBS. After 30 minutes of incubation, cell suspensions were
washed in PBS by 5-minute centrifugation and, for indirect
immunofluorescence procedures, reacted with the secondary anti-mouse
immunoglobulins in a 1:50 dilution, for 30 minutes. At the end of
incubations, cells were then centrifuged in PBS (1600 rpm, 5 minutes),
resuspended in 100 μml of PBS, and analyzed on a flow cytometer
(FACScan; Becton Dickinson, Meylon, France), according to previously
validated methods.715

Flow Cytometry Processing

The linear plot giving granulometry versus cell size consistently
revealed a single cell population (Fig. 1) . Analytic gates were set around this population to exclude cellular
debris and aggregates. The number of positive conjunctival cells was
then obtained from logarithmic cytograms of mean fluorescence
intensities. The superior level of fluorescence intensity obtained for
the isotypic control antibody was considered as the limit of background
fluorescence and the threshold of positivity for the tested antibodies.
In each sample, at least 1000 cells were analyzed. All specimens were
analyzed in a masked manner, in that the examiner did not know the
clinical history of patients.

Data were further expressed as a quantimetric assessment of
fluorescence intensities by using calibrated fluorospheres to translate
the mean fluorescence of each sample into standardized arbitrary
fluorescence units (AUFs), according to a previously published
method.15 A calibration curve was established during each
flow cytometric procedure by using four different beads (Immunobrite;
Coulter, Hyaleh, FL) with standardized fluorescence
intensities.17 This technique allowed quantification and
objective comparisons between days and therefore controlled the
reliability and quality of measurements. The real number of AUFs was
obtained by subtraction of the isotypic negative control from the total
AUFs calculated for each marker. The same flow cytometer was used
during the study. On a weekly basis, the machine was tested for
calibration by processing a set of calibrated beads used as controls.

Statistical comparisons were performed with the Mann–Whitney test and
the Z correlation test, at a 0.05 level of significance (Statview
IV for Windows; Abacus, Berkeley, CA).

Results

Study Population and Specimen Characteristics

Flow cytometry was performed on 243 IC samples collected before
randomization, which generated valid baseline data from 169 patients.
Seventy-four samples yielded invalid results for the following reasons:
insufficient sample—that is, number of cells too low or dry or wrong
membrane (n = 36), sample contamination by fluorescein
before collection (n = 6), patient withdrawal
(n = 5), transient machine breakdown (n = 9), and damaged cell samples discarded on the basis of the cell
distribution pattern after flow cytometry (n = 18). In
qualified specimens, homogeneous populations of conjunctival cells
could be collected, with few debris or aggregates (Fig. 1) . However,
because at least 1000 cells were required per analysis and a minimum of
seven analyses (five markers and two controls) were performed, the
number of cells in the sample was to be greater than 10,000, and poorer
or nonhomogeneous specimens were discarded. Range of cell numbers
collected by IC was therefore 10,000 to 685,000 cells per specimen that
reached the level of quality required for reliable analyses.

For the 169 patients with valid baseline samples, the mean (range) age
was 57.1 years (18–86 years) and 86.4% (146/169) were women. From
these 169 patients, 41% (70/169) had SS, according to the clinical and
biological criteria reported by Vitali et al.18(Table 2) . At baseline, in the overall population, mean Schirmer’s test was
1.47 mm at 5 minutes, corneal staining score was 2.96 (of a maximal
score of 5), and total nasal and temporal lissamine green staining
reached 5.47 (of a maximal score of 10). No difference was found
between the two populations of patients with and without SS in age, sex
ratio, or clinical data.

Flow Cytometry Results

Immunofluorescence flow cytometry at baseline, expressed in AUFs
and percentages of positive cells in individual specimens, are
summarized in Table 3 . Cytograms of HLA DR and CD40 expressions in a normal and a
KCS-affected eye are given in Figure 2 to show typical flow cytometric patterns. Of the 169 specimens
available for analysis, each antibody could be interpreted in 169, 164,
159, 168, and 168 samples for HLA DR, CD40, CD40 ligand, APO2.7, and
Fas, respectively.

HLA DR expression was found at positive AUF levels above the negative
control in all but three specimens. Mean percentage of HLA DR–positive
cells was 57% ± 32%, and mean fluorescence level was 102,973 ±
138,313 AUF (mean ± SD) with a maximal value of 774,254 AUF.
Results of HLA DR expression from the SS subgroup (Table 3) showed
significantly higher levels in AUFs than in patients without, but not
in the percentage of positive cells (131,021 ± 154,365 AUF versus
83,141 ± 122,713 AUF, P = 0.0039, Mann–Whitney
test). As expected, comparison of the overall population with normal
specimens showed highly significant differences, both in terms of
percentage of positive cells and in levels of expression
(P < 0.0001 compared with normal eyes for both
criteria; Fig. 3 ).

CD40 was found in all specimens (Table 3 , Figs. 2and 3 ). Percentage of
positive cells ranged from 14% to 100% with a mean percentage of
positive cells of 89% ± 14%. The CD40 fluorescence levels ranged
from 14,388 to 285,105 AUF with a mean level of 67,106 ± 37,092
AUF. In contrast, CD40 ligand was inconstantly found, in only 63% of
specimens, with a mean percentage of positive cells of 9% ± 15%. The
CD40 ligand fluorescence levels ranged from 0 to 50,442 AUF with a mean
of 6,503 ± 9,164 AUF. As observed for HLA DR, CD40 expression
from the SS subgroup showed a significantly higher level than in the
non-SS eyes with KCS (74,595 ± 36,429 AUF versus 61,529 ±
36,785 AUF, P = 0.0009), but not a significantly
different percentage of positive cells. CD40 ligand, however, did not
significantly differ in the two groups. Comparison of CD40 expression
with normal specimens also showed highly significant differences, both
in terms of percentage of positive cells and in quantified levels of
fluorescence (P < 0.0001 for the two criteria). CD40
ligand was also found significantly more expressed in eyes with KCS
than in normal ones (P = 0.007 in percentage of
positive cells; P = 0.02 in AUF).

Data from the apoptosis-related marker APO2.7 were available from 168
patients, all found positive. The range of percentage of positive cells
was 8% to 100% with a mean of 77% ± 25%, and a mean level of
expression of 6999 ± 6087 AUF. No difference was found between SS
and non-SS groups. Fas was also found on surface conjunctival cells in
all specimens. The analysis of Fas expression showed that most cells
were immunolabeled (95% ± 9%) with minimal and maximal values
respectively of 22% and 100%. Fas fluorescence levels ranged from
2,956 to 82,026 with a mean ± SD of 15,760 ± 10,224 AUF in
the overall population. Results of Fas expression from the SS group
showed significantly higher levels than in the non-SS group
(17,565 ± 11,037 AUF versus 14,038 ± 9878 AUF, P = 0.01). However, no significant difference could be
found between normal subjects and patients with KCS for Fas and APO2.7.

Highly significant positive correlation was found between HLA DR and
CD40, both in percentages of positive cells and in AUFs
(R2 = 0.37, P <
0.0001; and R2 = 0.31, P < 0.0001, respectively), between HLA DR and CD40
ligand percentages and AUF (R2 = 0.28, P = 0.0005; and R2 =
0.22, P = 0.005, respectively), between HLA DR and Fas,
only in AUF (R2 = 0.36, P < 0.0001). CD40 percentages and AUF were also
significantly correlated with CD40 ligand and Fas, but APO2.7 was not
significantly correlated with any of the other markers.

Discussion

The results from this analysis at baseline and comparisons with
normal patients as controls confirm that the ocular surface of patients
with moderate to severe KCS presents clear signs of inflammation and
imbalance of apoptosis-related receptors. SS is an autoimmune disease
involving not only the lachrymal glands but the whole ocular surface
and causing chronic inflammation, with lymphocytic infiltrates and
apoptosis of ocular epithelial cells. Even in non-SS dry eye, however,
an inflammatory reaction has been demonstrated in KCS345678 that could be hypothesized to result from chronic ocular surface
dryness and epithelial cell degeneration. As suggested by Stern et
al.,19 neural deregulation in the ocular surface and
lachrymal glands may result from continuous secretion of
proinflammatory cytokines and may exacerbate ocular surface damage by
decreasing the tearing reflex. The role of apoptosis in ocular cells
has also been shown in ocular surface disorders and in KCS; Fas and
APO2.7 were found to be significantly correlated to the expression of
inflammatory markers.15 In lachrymal glands of patients
with SS, acinar cells are infiltrated by CD8+ lymphocytes that adhere to acinar cells and induce epithelial apoptosis
by implying the Fas–Fas ligand pathway.20 Similarly, in
dog models of SS, apoptotic acinar cells and lymphocytes with decreased
levels of apoptosis can be observed within lachrymal glands. Lymphocyte
infiltration results in complete atrophy of lachrymal glands that may
be reversed by topical treatment with cyclosporine.21

High HLA DR expression by surface conjunctival epithelial cells was
thus found in this large series of patients with moderate to severe dry
eye. Almost all eyes showed high levels of HLA DR–positive epithelial
cells. These results correlated well, however, with those previously
reported using similar methods of flow cytometry in
impression715 or brush9 cytology. Class II
antigens HLA DR are membrane antigens expressed by immunocompetent
cells, normally restricted to antigen-presenting cells. In inflammatory
disorders, HLA DR expression may be induced in epithelial cells. HLA DR
has thus been reported to be overexpressed in the conjunctival
epithelium in chronic conjunctivitis and in dry
eyes.678922

In the present study, similar results were observed in SS and non-SS
eyes in patients with high levels of inflammatory and apoptotic
markers, although HLA DR, CD40, and Fas were expressed at significantly
higher levels in patients with SS. Tsubota et al.9 also
found very high HLA DR expression in patients with SS but much lower
levels in those with KCS without SS. The ocular surface in patients
with dry eye without SS may in their study have been less severely
injured than in the present work, in which all patients, whatever the
underlying diagnosis, had significant KCS, assessed by very low
Schirmer’s test results and intense corneal staining. This could
indicate that KCS per se may induce chronic inflammation in the ocular
surface by the chronic injury to epithelial cells caused by tear
deficiency, mechanical abrasion by the eyelid, and possibly the absence
of trophic factors, such as epidermal growth factor or transforming
growth factor beta.1823 Conversely, it may be
hypothesized that proinflammatory mediators chronically liberated on
the ocular surface cause epithelial cells to degenerate over time,
because various cytokines may stimulate apoptosis in many cell types.
Such mediators also may cause degeneration by interfering with neural
connections that regulate ocular surface homeostasis.19

Strong relationships between inflammation and apoptosis have therefore
been demonstrated in various epithelial cells and especially on ocular
surface cells. Fas is normally expressed by conjunctival epithelial
cells, in which it is positively correlated with levels of HLA DR
antigens, and stimulating anti-Fas antibodies causes epithelial cell
apoptosis.15 Moreover, interferon-γ has been shown to
increase expression of Fas and HLA DR by conjunctival
cells24 and to induce apoptosis, by different metabolic
pathways, including stimulation of the Fas system.2425 In
the present study, we confirmed the significant correlation between HLA
DR and Fas in patients with dry eye, with or without SS. We did not
find in this population, however, the overexpression of the apoptotic
marker APO2.7, as previously demonstrated in ocular surface
disorders.15 This could be related to the low levels of
expression obtained when using direct immunofluorescence procedures,
which reduce the possibility for slight differences to be raised to
significance. We cannot exclude, however, the hypothesis that in the
most severe cases of KCS, tissue differentiation may have been
impaired, as shown by Jones et al.23 in SS, resulting in
an increased number of epithelial layers and possibly a low level of
apoptosis in the superficial layers.

CD40 and CD40 ligand were also found in ocular surface cells of
patients with KCS, at upregulated levels in dry eyes compared with
normal eyes, and at higher levels in eyes with than in eyes without SS.
CD40 was also positively correlated with HLA DR, CD40 ligand and Fas
expressions. CD40 upregulation may be observed in inflammatory
conditions in various tissues and cell systems.26 CD40 has
been shown to be involved in lymphocyte stimulation, chronic
inflammation, and apoptosis.2627 As HLA DR, its
expression in epithelial cells is stimulated by various cytokines, such
as interferon-γ or TNF-α,28 which could be synthesized
in the ocular surface and lachrymal glands by infiltrating lymphocytes.
CD40 has also been shown in other systems to interfere with the
Fas–Fas ligand pathway.29 However, CD40–CD40 ligand
interaction alone could not be sufficient to provide a mitogenic signal
to T cells and should rather be implicated in amplifying the
inflammatory reaction.30

Mechanisms of such overexpressions of inflammatory markers may
therefore involve such cytokines as interferon-γ and
TNF-α.92431 These two cytokines have a synergistic
effect on HLA DR expression by conjunctival epithelial
cells.9 Interferon-γ also stimulates Fas expression,
Fas-induced apoptosis and CD40 expression in a conjunctival cell
line.1624 Whether HLA DR–expressing conjunctival
epithelial cells acquire antigen-presenting properties, as do corneal
epithelial31 and lachrymal acinar cells,5 remains to be determined. However, it may be hypothesized that
immunologically activated epithelial cells can be targeted by
lymphocytes in cytotoxic reactions920 and/or that they
participate in recruitment of inflammatory cells.

Nevertheless, overexpression of inflammation- and apoptosis-related
antigens confirms that epithelial cells, even in nonautoimmune KCS, are
directly involved in the inflammatory process. These data provide
additional rationale for using cyclosporine ophthalmic emulsion in KCS,
whatever its origin, to reduce both inflammatory and apoptotic
involvement of ocular surface cells. Flow cytometry provided a valuable
tool to reliably assess and quantify the level of inflammatory
impairment of conjunctival epithelial cells, both at baseline, and
throughout the study, to monitor the immunomodulatory effect of
cyclosporine. Based on the present study, analyses of eyes receiving
the masked treatments in this large European multicenter trial on
Cyclosporin A Ophthalmic Emulsion, will further be presented and will
provide major information concerning the effect of this drug on the
ocular surface in moderate to severe KCS.

Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1999.

Flow cytometry cytogram showing the conjunctival cell population
obtained by IC. Cell population appears homogeneous, with few
aggregates (top and right side), and
small size events corresponding to cell debris
(arrows).

Figure 1.

Flow cytometry cytogram showing the conjunctival cell population
obtained by IC. Cell population appears homogeneous, with few
aggregates (top and right side), and
small size events corresponding to cell debris
(arrows).

Vitali C, Moutsopoulos HM, Bombardieri S. The European Community Study Group on diagnostic criteria for Sjogren’s syndrome: sensitivity and specificity of tests for ocular and oral involvement in Sjogren’s syndrome. Ann Rheum Dis. 1994;53:637–647.[CrossRef][PubMed]

Flow cytometry cytogram showing the conjunctival cell population
obtained by IC. Cell population appears homogeneous, with few
aggregates (top and right side), and
small size events corresponding to cell debris
(arrows).

Figure 1.

Flow cytometry cytogram showing the conjunctival cell population
obtained by IC. Cell population appears homogeneous, with few
aggregates (top and right side), and
small size events corresponding to cell debris
(arrows).